Controlled exchange of protein and RNA between the nucleus and cytoplasm is a process of fundamental importance to all eukaryotic cells and essential for proper gene expression. One of the most outstanding questions in the nuclear transport field involves delineating how messenger RNAs (mRNAs) are exported through nuclear pore complexes (NPCs) embedded in the nuclear envelope. It is also unclear how the mRNA export mechanism is connected to translation regulation. The long-term goal of this project is to elucidate the molecular sequence of events required for coupling mRNA cargo translocation through NPCs to cytoplasmic mRNA trafficking and translation. We hypothesize that key events for mRNA export directionality are controlled at the NPC by the action of multiple factors. We further speculate that some of the NPC associated factors are multi-functional and effectively link the export and cytoplasmic translation steps. To test these hypotheses, we propose three specific aims. In aims one and two, we will use the S. cerevisiae model to study this highly conserved machinery. In aim one, we will define the mechanism by which mRNA cargo translocates through the NPC. This involves direct interactions between the mRNA transport receptor Mex67-Mtr2 and critical binding sites in NPC proteins termed FG repeats. A panel of novel FG repeat mutants will be used to investigate the precise requirements for NPC translocation. A combination of genetic, microscopy and biochemistry approaches will be used to define key events at the NPC cytoplasmic face. We will determine how the essential mRNA export factor Gle1 and inositol hexakisphosphate (IP6) activate the DEAD-box protein Dbp5 for targeted changes in the mRNA-protein complex and directional export. The second aim will investigate how mRNA export and translation initiation/termination are functionally linked by the actions of Gle1, IP6, and Dbp5. We will generate allele specific gle1 mutants, and test for Dbp5-mediated remodeling of translation termination complexes. To reveal how translation initiation is controlled, genetic and biochemical assays will be conducted to analyze the mechanism by which Gle1 controls an initiation-specific DEAD-box protein. In aim three, we will extend these studies to mammalian cells and identify the molecular determinants for Gle1 dysfunction in disease. The mRNA export and translation perturbations will be measured for a human GLE1 mutant (gle1-FINmajor) that is causally linked to a lethal fetal motor neuron disease. Direct protein-protein interactions that are altered by the gle1-FINmajor mutant will also be identified. Together, these studies will define the machinery controlling critical steps in the mRNA life cycle, and impact our understanding of the role for altered mRNA transport and metabolism in human disease. PUBLIC HEALTH RELEVANCE: PROJECT NARRATIVE Understanding how proteins and RNA move between the nucleus and cytoplasm will provide novel insights into the pathology of multiple human diseases. Nuclear transport factors and the trafficking mechanism are altered in some cancers and viral infections, and dysfunction induced by inherited mutations in these factors is implicated in human developmental defects, heart disease, and immune system function. These studies will also provide critical knowledge for the ultimate identification and analysis of therapeutic targets.